Stem Cells and Genome Engineering

Dr. Shin-Il Kim and his lab mates have been working like machines to unravel a reprogramming mystery. Employing piggyBac transposons to deliver two seemingly similar polycistronic reprogramming cassettes (both employing the Yamanaka Factors Oct3/4, Sox2, Klf4, and c-Myc), achieves two drastically distinct reprogramming outcomes. With one vector, colonies are plentiful, yet ultimately fail to fully reprogram. With the other, colonies are few but true – with most going on to activate a Nanog-GFP pluripotency reporter.

So, why the difference? Looking down to the sequence level, we found that the Klf4 open reading frame in the two vectors differed subtly in length. In the vector which produced primarily partially reprogrammed cells, Klf4 was truncated by 9 N-terminal amino acids. Amazingly, replacement of these 9 amino acids suppressed colony formation and recovered the reprogramming capacity of the deficient vector.

These phenotypes are ultimately achieved by modulating the relative factor stoichiometry, where short Klf4 produces less protein than long. Interestingly, this affect appears to be dependent on 2A-peptide linkage, as monocistronic short and long Klf4 function nearly indistinguishably. Importantly, this modulation of stoichiometry is reflected in global gene expression changes during reprogramming, highlighting difficulties in direct comparison of gene expression profiles from distinct reprogramming systems.